Matrix-assisted laser desorption ionization (MALDI) mass spectrometry, electrospray ionization mass spectrometry (ESI/MS), tandem mass spectrometry (MS/MS), on-line liquid chromatography-mass spectrometry (LC/MS), combined capillary electrophoresis (CE) and mass spectrometry, and accurate mass measurement are the techniques of current interest. The off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) and with CE is under investigation as an alternate approach for the analysis of complex biological and synthetic mixtures. This approach involves automatically collecting and spotting the HPLC eluent on a MALDI sample plate for subsequent MS analysis, which can include accurate mass measurement and post-source decay fragmentation analysis or MS/MS. An advantage of this approach over on-line LC/MS is the generation of an archive of the original separated sample for reanalysis. A similar approach is being applied with CE where on-linesample concentration techniques involving electrophoretic focusing of large-volume sample injections prior to peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS. Our goal is to automate this process. Preliminary studies of MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix show promise for the rapid analysis of small molecules.Synthetic derivatives built on a constrained diacylated glycerol scaffold (DAG-lactones) have been identified as potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents comprising R1 and R2, these DAG-lactones appear to have some degree of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic libraries be rapidly characterized and their structures established before biological evaluation. One of the rapid mass spectral approaches employed for the initial characterization of the larger synthetic DAG-lactone libraries is FAB/MS mixture analysis. Analysis of DAG-lactone mixtures correlates directly with individual analysis, substantially reduces the number of samples to be examined, results in enhanced analytical turn-around and is amenable to accurate mass measurement. Flow-injection ESI/MS is not suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. Flow injection-atmospheric pressure chemical ionization produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach appears to be complementary to FAB/MS in terms of spectral information. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries although variable alkali metal ion cationization complicates spectral interpretation. Matrix-less MALDI is currently under investigation as an approach to resolve this problem and provide an even more rapid analysis approach. Our goal is the structural characterization of all 96 library components in one day.